In various medical and chemical tests it is often desirable to determine the occurrence of nonoccurrence of a reaction in a fluid sample. For example, in the medical field it is often highly desirable to predetermine the compatibitlity of donor tissues with host tissue prior to an organ transplant. Presently, determinations of tissue types are accomplished using cytotoxicty assays. This involves reacting cells from the donor (and in a separate determination, the host) with antisera directed against specific cell surface antigens, known as histocompatibility antigens, in the presence of a source of complement. Binding of antibody to antigen on the cell surface leads to complement-mediated lysis of the cell. If a vital dye is also present in the test medium, it is possible to distinguish live from dead cells based on their staining. Thus, it is possible to determine the repertoire of histocompatibility antigens which a particular individual's cells express. A technician visually inspects each well in the plate to determine the extent of reaction as measured by cell death. The reading and interpretation of such a plate requires about ten minutes to accomplish, depending on the number of wells in the plate and the skill of the reader.
Recent developments in molecular biology have provided new techniques in which the extent of an antigen-antibody reaction can be determined by the formation of a colored product in the fluid sample. The resulting coloration of the fluid is significantly easier to read and much less subjective than the aforementioned procedure in which cell viability is evaluated by staining with a vital dye. The new technique also gives a quantitative measure of the strength of the reaction while the previously used method yielded semi-quantitive information, at best. This improved technique affords an opportunity to automate the reading of multi-well plates by using vertical photometric density measurements. However, due to the small size and geometry of the wells within which the fluid sample is contained, a variety of problems have heretofore prevented the automation of this process.
The first problem is caused by the small diameter of the microwells presently available in microwell plates. A typical microwell in a Terasaki plate has an inverted frusto-conic shape. The bottom (narrowest) portion of the well has a substantially transparent window with a diameter of only approximately 0.047 inch. The open top (widest portion) of the well has a typical diameter of only approximately 0.16 inch. Therefore, a substantial fluid meniscus is typically formed on the top surface of the fluid sample in the well. The meniscus can vary in curvature from one well to the next thereby frustrating attempts to optically compensate for the refractive effect of the the meniscus on an interrogating light beam. The accuracy of an optical density measurement depends on providing a repeatably accurate light beam path through the fluid sample to a detector. A fundamental presumption in a density measurement of this type is that light not received at the detector end of the system is absorbed by the fluid. Stray light beams, such as caused when refracted by a meniscus, are incorrectly read by such a system as having been absorbed by the fluid. An inaccurate measurement therefore results. Attempts to predict the position and curvature of the meniscus to optically compensate for the refractive effect of the meniscus are frustrated by the variability of the meniscus curvature and position within the microwell.
A second significant problem in the automation of density measurements in microwell plates relating to the small size of the microwell is the ability to accurately place the 0.047 inch diametr well bottom directly beneath an interrogating beam of light. Typically, the plates containing the wells are mass produced in a plastic molding process. The wells are not always perfectly centered on their respective matrix positions. There is also a significant variation of the position of the matrix itself relative to the sidewalls of the plate.
A third significant problem encountered with modern microwell designs results from the irregularity of the plastic surface in the well bottom which can also refract a light beam passing therethrough.
The present invention solves the above heretofore unsolved problems in an automated system for measuring the optical density of a fluid sample in microwell plates.